Response Of Human Mesenchymal Stem Cells Research Articles

Guideline for design of substrate stiffness for mesenchymal stem cell culture based on heterogeneity of YAP and RUNX2 responses.

Mesenchymal stem cells (MSCs) have the potential for self-renewal and multipotency to differentiate into various lineages. Thus, they are of great interest in regenerative medicine as a cell source for tissue engineering. Substrate stiffness is one of the most extensively studied exogenous physical factors; however, consistent results have not always been reported for controlling MSCs. Conventionally used stiff culture substrates, such as tissue-culture polystyrene and glass, enhance nuclear localization of a mechanotransducer YAP and a pre-osteogenic transcription factor RUNX2, and bias MSCs towards the osteogenic lineage, even without osteogenic-inducing soluble factors. The mechanosensitive nature and intrinsic heterogeneity present challenges for obtaining reproducible results. This review summarizes the heterogeneity in human MSC response, specifically, nuclear/cytoplasmic localization changes in the mechanotransducer yes-associated protein (YAP) and the osteogenic transcription factor RUNX2, in response to substrate stiffness. In addition, a perspective on the intracellular factors attributed to response heterogeneity is discussed. The optimal range of stiffness parameters, Young's modulus, for MSC expansion culture to suppress osteogenic differentiation bias through the suppression of YAP and RUNX2 nuclear localization, and cell cycle progression is likely to be surprisingly narrow for a cell population from an identical donor and vary among cell populations from different donors. We believe that characterization of the heterogeneity of MSCs and understanding their biological meaning is an exciting research direction to establish guidelines for the design of culture substrates for the sophisticated control of MSC properties.

The Implication of Spatial Statistics in Human Mesenchymal Stem Cell Response to Nanotubular Architectures.

IntroductionIn recent years there has been ample interest in nanoscale modifications of synthetic biomaterials to understand fundamental aspects of cell-surface interactions towards improved biological outcomes. In this study, we aimed at closing in on the effects of nanotubular TiO2 surfaces with variable nanotopography on the response on human mesenchymal stem cells (hMSCs). Although the influence of TiO2 nanotubes on the cellular response, and in particular on hMSC activity, has already been addressed in the past, previous studies overlooked critical morphological, structural and physical aspects that go beyond the simple nanotube diameter, such as spatial statistics.MethodsTo bridge this gap, we implemented an extensive characterization of nanotubular surfaces generated by anodization of titanium with a focus on spatial structural variables including eccentricity, nearest neighbour distance (NND) and Voronoi entropy, and associated them to the hMSC response. In addition, we assessed the biological potential of a two-tiered honeycomb nanoarchitecture, which allowed the detection of combinatory effects that this hierarchical structure has on stem cells with respect to conventional nanotubular designs. We have combined experimental techniques, ranging from Scanning Electron (SEM) and Atomic Force (AFM) microscopy to Raman spectroscopy, with computational simulations to characterize and model nanotubular surfaces. We evaluated the cell response at 6 hrs, 1 and 2 days by fluorescence microscopy, as well as bone mineral deposition by Raman spectroscopy, demonstrating substrate-induced differential biological cueing at both the short- and long-term.ResultsOur work demonstrates that the nanotube diameter is not sufficient to comprehensively characterize nanotubular surfaces and equally important parameters, such as eccentricity and wall thickness, ought to be included since they all contribute to the overall spatial disorder which, in turn, dictates the overall bioactive potential. We have also demonstrated that nanotubular surfaces affect the quality of bone mineral deposited by differentiated stem cells. Lastly, we closed in on the integrated effects exerted by the superimposition of two dissimilar nanotubular arrays in the honeycomb architecture.DiscussionThis work delineates a novel approach for the characterization of TiO2 nanotubes which supports the incorporation of critical spatial structural aspects that have been overlooked in previous research. This is a crucial aspect to interpret cellular behaviour on nanotubular substrates. Consequently, we anticipate that this strategy will contribute to the unification of studies focused on the use of such powerful nanostructured surfaces not only for biomedical applications but also in other technology fields, such as catalysis.

Human Mesenchymal Stem Cell Response to Lactoferrin-based Composite Coatings.

The potential of mesenchymal stem cells (MSCs) for implantology and cell-based therapy represents one of the major ongoing research subjects within the last decades. In bone regeneration applications, the various environmental factors including bioactive compounds such as growth factors, chemicals and physical characteristics of biointerfaces are the key factors in controlling and regulating osteogenic differentiation from MSCs. In our study, we have investigated the influence of Lactoferrin (Lf) and Hydroxyapatite (HA) embedded within a biodegradable PEG-PCL copolymer on the osteogenic fate of MSCs, previous studies revealing an anti-inflammatory potential of the coating and osteogenic differentiation of murine pre-osteoblast cells. The copolymer matrix was obtained by the Matrix Assisted Pulsed Laser Evaporation technique (MAPLE) and the composite layers containing the bioactive compounds (Lf, HA, and Lf-HA) were characterised by Scanning Electron Microscopy and Atomic Force Microscopy. Energy-dispersive X-ray spectroscopy contact angle and surface energy of the analysed coatings were also measured. The characteristics of the composite surfaces were correlated with the viability, proliferation, and morphology of human MSCs (hMSCs) cultured on the developed coatings. All surfaces were found not to exhibit toxicity, as confirmed by the LIVE/DEAD assay. The Lf-HA composite exhibited an increase in osteogenic differentiation of hMSCs, results supported by alkaline phosphatase and mineralisation assays. This is the first report of the capacity of biodegradable composite layers containing Lf to induce osteogenic differentiation from hMSCs, a property revealing its potential for application in bone regeneration.

Human Bone Marrow-Derived Mesenchymal Stem Cells Home via the PI3K-Akt, MAPK, and Jak/Stat Signaling Pathways in Response to Platelet-Derived Growth Factor.

Mesenchymal stem cells (MSCs) have great potential to improve clinical outcomes for many inflammatory and degenerative diseases either through intravenously delivered MSCs or through mobilization and migration of endogenous MSCs to injury sites, termed "stem cell homing." Stem cell homing involves the processes of attachment to and transmigration through endothelial cells lining the vasculature and migration through the tissue stroma to a site of injury or inflammation. Although the process of leukocyte transendothelial migration (TEM) is well understood, far less is known about stem cell homing. In this study, a transwell-based model was developed to monitor adherence and TEM of human MSCs in response to chemokine exposure. Specifically, transwell membranes lined with human synovial microvascular endothelial cells were partitioned from the tissue injury-mimetic site containing chemokine stromal cell-derived factor-1 (SDF-1). Two population subsets of MSCs were studied: migratory cells that initiated transmigration on the endothelial lining and nonmigratory cells. We hypothesized that cells would adhere to and migrate through the endothelial lining in response to SDF-1 exposure and that gene and protein expression changes would be observed between migratory and nonmigratory cells. We validated a vasculature model for MSC transmigration that showed increased expression of several genes and activation of proteins of the PI3K-Akt, MAPK, and Jak/Stat signaling pathways. These findings showed that MSC homing may be driven by activation of PDGFRA/PI3K/Akt, PDGFRA/MAPK/Grb2, and PDGFRA/Jak2/Stat signaling, as a result of SDF-1-stimulated endothelial cell production of platelet-derived growth factor. This model can be used to further investigate these key regulatory molecules toward the development of targeted therapies.

Silk fibroin scaffolds with inverse opal structure for bone tissue engineering.

How scaffold porosity, pore diameter and geometry influence cellular behavior is‐although heavily researched ‐ merely understood, especially in 3D. This is mainly caused by a lack of suitable, reproducible scaffold fabrication methods, with processes such as gas foaming, lyophilization or particulate leaching still being the standard. Here we propose a method to generate highly porous silk fibroin scaffolds with monodisperse spherical pores, namely inverse opals, and study their effect on cell behavior. These silk fibroin inverse opal scaffolds were compared to salt‐leached silk fibroin scaffolds in terms of human mesenchymal stem cell response upon osteogenic differentiation signals. While cell number remained similar on both scaffold types, extracellular matrix mineralization nearly doubled on the newly developed scaffolds, suggesting a positive effect on cell differentiation. By using the very same material with comparable average pore diameters, this increase in mineral content can be attributed to either the differences in pore diameter distribution or the pore geometry. Although the exact mechanisms leading to enhanced mineralization in inverse opals are not yet fully understood, our results indicate that control over pore geometry alone can have a major impact on the bioactivity of a scaffold toward stem cell differentiation into bone tissue. © 2016 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2074–2084, 2017.

Human Mesenchymal Stem Cells Response to Magnesium-based Biomaterials

Implants have been used as replacement materials for lost tissues and are mainly from metals such as stainless steel and titanium. Metals implants may release toxic elements, and alternative such as magnesium-based implants are suggested but its high degradation rate in human bio-environment limit its uses. Thus, we proposed alloying the magnesium with zinc atoms and dispersing bioactive hydroxyapatite (HAp) within the magnesium and zinc matrix and assess the effects of the conditioned-medium from the Mg-Zn-HAP on human bone marrow mesenchymal stem cells (hMSC) viability. The Mg with and without Zinc powders were purchased from Merck and Alfa Aesar. The precursor powder has particle size of 0.06-0.3mm. Powder mixtures with different composition i.e., 100 weight percent (wt%) Mg, 9:1 Mg-Zn, 90:9:1 Mg-Zn-Mn, 9:1 Mg-Zn + 10 wt% HAp and 90:9:1 Mg-Zn-Mn + 10 wt% HAP were sintered at 300°C. Then, the powder was incubated with culture medium (1.0mg/ml and 2.0mg/ml) and placed in an incubator shaker for 4hours. The medium was filtered using 0.2μm syringe filter and kept at 4°C. The conditioned-medium was supplemented with 10% (v/v) fetal bovine serum and 1% Pen/Strep and incubated overnight at 37°C in a CO2 incubator prior to use on hMSC. The cell viability was assessed using Alamar Blue assays at Days 1, 2, 4 and 7. The hMSC showed increased proliferation when cultured in MgZn- and MgZnMn-conditioned medium when compared to Mg-HAp conditioned medium. These metallic ions may play role in stimulating cell proliferation. The presence of metallic ions such as Mg, Mn and Zn promote hMSC proliferation and may play role as an important elements for regeneration of hard tissue during implantation.

Substance P enhances the proliferation and migration potential of murine bone marrow-derived mesenchymal stem cell-like cell lines.

Due to the therapeutic characteristics of bone marrow (BM)-derived mesenchymal stem cells (MSCs), clinical trials are testing the use of autologous or allogeneic MSCs for the treatment of several conditions. These therapies require large numbers of MSCs and numerous studies are attempting to find substances that could enhance the egression of endogenous MSCs from the BM into the periphery and increase their proliferation in vivo and in vitro. It has been reported that substance P (SP) has the potential to increase the expansion of MSCs in vivo and to induce their mobilization from the BM into the periphery. The aim of the present study was to investigate the effects of SP on the migration and proliferation potential of two BM-derived MSC-like cell lines, ST2 and OP9. SP was found to induce the migration potential of ST2 cells in vitro. Furthermore, SP increased the proliferation of the MSCs cell line, OP9 cell line. Cyclin D1 expression was observed to increase in the OP9 cells, indicating the activation of the cell cycle in response to SP. The upstream signals involved in these phenomena have yet to be elucidated, although previous studies have proposed the activation of the extracellular signal-regulated kinase-1/2 and Wingless/β-catenin pathways as possible mediators of the cellular proliferation of human MSCs in response to SP. The present results therefore suggest that SP would facilitate the obtainment of higher numbers of endogenous MSCs from patients or donors and/or shorten the process of in vitro expansion that could cause the MSCs to undergo changes in their innate therapeutic characteristics prior to their use in therapy.

A comparative analysis of human mesenchymal stem cell response to hypoxia in vitro: Implications to translational strategies

PurposeMesenchymal stem cells (MSCs) are particularly valuable for structural tissue replacement. We compared the response to hypoxia among human MSCs derived from four different clinically relevant sources as an adjunct to translational developments. MethodsImmunophenotypically indistinguishable human MSC lineages derived from bone marrow (bmMSCs), adipose tissue (adMSCs), amniotic fluid (afMSCs), and umbilical cord blood (cbMSCs) were submitted to either room air or 1% O2, under otherwise standard culture conditions. Cell expansion and quantitative RT-PCR data were obtained at different time points. Statistical analysis was by two-way mixed model and the F-test (P<0.05). ResultsThe effect of hypoxia on expansion kinetics was dependent on cell source. Only prenatal sources of MSCs – afMSCs (P=0.002) and cbMSCs (P<0.001) – proliferated significantly faster under hypoxia than normoxia. Increased HIF1-alpha expression correlated consistently with increased cell expansion only among afMSCs. There were no significant variabilities in Survivin, Oct-4, and VEGF expressions. ConclusionsMesenchymal stem cell tolerance to hypoxia in vitro varies with cell source. Prenatal cells, particularly those derived from amniotic fluid, are more robust than their postnatal counterparts. HIF1-alpha may play a role in the amniotic fluid-derived cells’ enhanced response. These findings should inform the choice of mesenchymal stem cells for prospective regenerative strategies.

Human mesenchymal stem cells response to multi-doped silicon-strontium calcium phosphate coatings

The search for apatitic calcium phosphate coatings to improve implants osteointegration is, nowadays, preferentially focused in the obtaining of compositions closer to that of the inorganic phase of bone. Silicon and strontium are both present in trace concentrations in natural bone and have been demonstrated, by separate, to significantly improve osteoblastic response on calcium phosphate bioceramics. This work aims the controlled and simultaneous multi-doping of carbonated calcium phosphate coatings with both elements, Si and Sr, by pulsed laser deposition technique and the biological response of human mesenchymal stem cells to them. A complete physicochemical characterization has been also performed to analyze the coatings and significant positive effect was obtained at the osteogenic differentiation of cells, confirming the enormous potential of this multi-doping coating approach.

Human Mesenchymal Stem Cell Response to 444 Ferritic Stainless Steel Networks

ABSTRACTThe aim of this work is to improve bone-implant bonding. This can, potentially, be achieved through the use of an implant coating composed of fibre networks. It is hypothesised that such an implant can achieve strong peri-prosthetic bone anchorage, when seeded with human mesenchymal stem cells (hMSCs). The materials employed were 444 and 316L stainless steel fibre networks of the same fibre volume fraction. The present work confirms that hMSCs are able to proliferate and differentiate towards the osteogenic lineage when seeded onto the fibre networks. Cellular viability, proliferation and metabolic activity were assessed and the results suggest higher proliferation rates when hMSC are seeded onto the 444 networks as compared to 316L. Cell distribution was found uniform across the seeded surfaces with 444 showing a somewhat higher infiltration depth.

Cellular and transcriptomic analysis of human mesenchymal stem cell response to plasma-activated hydroxyapatite coating

Atmospheric pressure plasma has recently emerged as a technique with a promising future in the medical field. In this work we used the technique as a post-deposition modification process as a means to activate hydroxyapatite (HA) coatings. Contact angle goniometry, optical profilometry, scanning electron microscopy morphology imaging and X-ray photoelectron spectroscopy analysis demonstrate that surface wettability is improved after treatment, without inducing any concomitant damage to the coating. The protein adsorption pattern has been found to be preferable for MSC, and this may result in greater cell attachment and adhesion to plasma-activated HA than to untreated samples. Cell cycle distribution analysis using flow cytometry reveals a faster transition from G1 to S phase, thus leading to a faster cell proliferation rate on plasma-activated HA. This indicates that the improvement in surface wettability independently enhances cell attachment and cell proliferation, which is possibly mediated by FAK phosphorylation. Pathway-specific polymerase chain reaction arrays revealed that wettability has a substantial influence on gene expression during osteogenic differentiation of human MSC. Plasma-activated HA tends to enhance this process by systemically deregulating multiple genes. In addition, the majority of these deregulated genes had been appropriately translated, as confirmed by ELISA protein quantification. Lastly, alizarin red staining showed that plasma-activated HA is capable of improving mineralization for up to 3weeks of in vitro culture. It was concluded from this study that atmospheric pressure plasma is a potent tool for modifying the biological function of a material without causing thermal damage, such that adhesion molecules and drugs might be deposited on the original coating to improve performance.

Effect of RGD peptide coating of implant titanium surface on human mesenchymal stem cell response

연구 목적: 양극 산화 티타늄 임플란트의 표면에RGD펩타이드를 화학적 고정 및 물리적 흡착 방법을 통해 코팅하고, 이러한 코팅방법에 따른 표면 변화와 펩타이드의 코팅여부, 인간간엽줄기세포 배양시의 부착, 증식, 분화를 비교하여, 펩타이드를 임플란트 표면에 코팅시키는 방법과 세포의 반응 간의 관계를 분석하고자 하였다. 연구 재료 및 방법: 직경 12.0 mm, 두께 3.0 mm의 양극 산화 티타늄 디스크 상에, 대조군은 아무런 코팅을 시행하지 않았으며, 실험군은 표면에 형광 물질이 고정되어 있는 RGD펩타이드를 화학적 고정 방법과 물리적 흡착 방법으로 코팅시켰다. 펩타이드 코팅 이후의 표면 변화를 살펴보기 위해 주사전자현미경관찰, 형광현미경 관찰, X-ray Photoelectron Spectrometry (XPS) 분석을 시행하였다. 세포 부착 정도와 형태의 변화 및 증식 정도를 평가하였다. 분화의 정도를 살펴보기 위해, 정량중합효소연쇄반응, alkaline phosphatase activity assay, alizarin red assay를 이용하여 각각 분석하였다. 통계 분석은 SPSS (ver. 17.0, SPSS, IL, USA)프로그램을 이용하여 Kruskal-Wallis test로 유의성을 검증하였고, 사후 검정은 Bonferroni test를 시행하였다(P=.05). 결과: 형광 현미경, XPS 분석 결과, 두 가지 코팅 방법에서 모두 펩타이드의 코팅이 확인되었으며, 물리적 흡착 방법이 화학적 고정 방법보다 더 많은 양의 펩타이드를 코팅시킬 수 있었다. 코팅 방법의 차이에 따른 세포의 초기 부착 정도와 형태 변화, 증식의 정도에는 유의할만한 차이가 나타나지 않았다(P>.05). 세포의 분화 정도는 물리적 흡착 실험군에서 대조군과 화학적 흡착 실험군에서보다 collagen type I과 osteocalcin, osteopontin의 양이 증가되었으며, ALP activity가 유의하게 증가되었다(P 【Purpose: The aim of this in vitro study was to estimate surface characteristic after peptide coating and investigate biological response of human mesenchymal stem cell to anodized titanium discs coated with RGD peptide by physical adhesion and chemical fixation. Materials and methods: Fluorescence isothiocyanate (FITC) modified RGD-peptide was coated on the anodized titanium discs (diameter 12 mm, height 3 mm) using two methods. One was physical adhesion method and the other was chemical fixation method. Physical adhesion was performed by dip and dry procedure, chemical fixation was performed by covalent bond via silanization. In this study, human mesenchymal stem cell was used for experiments. The experiments consisted of surface characteristic evaluation after peptide coating, analysis about cell adhesion, proliferation, differentiation, and mineralization. Obtained data are statistically treated using Kruskal-Wallis test and Bonferroni test was performed as post hoc test (P=.05). Results: The evaluation of FE-SEM images revealed no diffenrence at micro-surfaces between each groups. Total coating dose was higher at physical adhesion experimental group than at chemical fixation experimental group. In cell adhesion and proliferation, RGD peptide coating did not show a statistical significance compared with control group (P>.05). In cell differentiation and mineralization, physical adhesion method displayed significantly increased levels compared with control group and chemical fixation method (P】

Evaluation of human mesenchymal stem cells response to biomimetic bioglass‐collagen‐hyaluronic acid‐phosphatidylserine composite scaffolds for bone tissue engineering

The aim of this study was to examine in vitro the response of human mesenchymal stem cells (hMSCs) on the novel biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine (BG-COL-HYA-PS) composite scaffold for potential use in bone tissue engineering. The initial attachment, the proliferation, migration and differentiation behavior of the cells on the BG-COL-HYA-PS composites were assessed in comparison with those on pure 58sBG, BG-COL, and BG-COL-HYA composites in either growth medium (L-DMEM supplemented with 10% fetal bovine serum) or osteogenic medium (growth medium supplemented with 0.1 microM dexamethasone, 10 mM beta-glycerophosphate, and 50 microM ascorbic acid). HMSCs attached, and subsequently proliferated and migrated on the BG-COL-HYA-PS composites to a significantly higher degree. The alkaline phosphatase (ALP) staining, ALP activity and the expression of the bone associated gene ALP, osteocalcin (OC), and osteopontin (OPN) was also significantly higher in the hMSCs on the BG-COL-HYA-PS scaffolds than those on the BG-COL, BG-COL-HYA composites and the pure 58sBG. These findings suggest that the BG-COL-HYA-PS composite porous scaffolds have high potential for use as scaffolds in bone tissue engineering and repair.